The mammalian central nervous system is a complex neuronal network consisting of a diverse array of cellular subtypes generated in a precise spatial and temporal pattern throughout development. Each neuronal subtype within a particular region of the brain and spinal cord carries a unique set of neurotransmitters and establishes connections with its own targets. It is the diversity in molecular and morphological characteristics of neurons which underlies neural circuit formation.
Extrinsic signals provide neuronal progenitors in the forming neural tube with positional identity, such that distinct types of neuronal progenitors express a unique combination of transcription factors. This transcriptional code determines neural progenitor identity. As progenitors differentiate, they generate distinct neuronal subtypes that are also characterized by transcriptional codes and secretion of specific transmitters. For example, motor neurons (MNs) are a highly specialized class of neurons that reside in the spinal cord and project axons in organized and discrete patterns to muscles to control their activity. Motor neurons secrete the transmitter acetylcholine, express transcription factors including MNX1 (also known as HB9), ISL1, and LHX3, and are derived from motor neuron progenitors which express the basic helix-loop-helix (bHLH) transcription factor OLIG2. During neurogenesis, OLIG2 is expressed by MNPcells and is required for the generation of MNs, while the homeodomain protein NKX2.2 is expressed in p3 progenitors and induces V3 neurons. Dessaud et al., Development 135:2489-2503 (2008). The most prominent MN diseases are spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS), in which MNs perish in the disease. For review, see Kanning et al., Annu. Rev. Neurosci. 33:409-410 (2010). Similarly, hindbrain serotonin neuronal progenitors express NKX2.2 together with GATA2 but not OLIG2 or PHOX2b and generate serotonin-secreting neurons that project to the entire brain and spinal cord. Numerous psychiatric disorders involve dysfunctional serotonin neurons. For review, see Gordis & Rohrer, Nat. Rev. Neurosci. Vol. 3(7):531-541 (2002); Kiyasova & Gaspar, Eur. J. Neurosci. Vol. 34(10):1553-1562, (2011).
Neural progenitor cells have been expanded in culture in the presence of mitogens such as epidermal growth factor (EGF) and/or fibroblast growth factor 2 (FGF2). For review, see Weiss et al., Trends Neurosci. Vol. 19:387-393 (1996). Neural progenitors expanded under such conditions exhibit diminished potential for generating neurons over glial cells. See Temple, Nature Vol. 414:112-117 (2001). This trend is in general agreement with the shift from neurogenesis to gliogenesis observed during normal development. Embryonic ventral mesencephalic progenitors, which produce robust dopaminergic neurons at the time of isolation, lose their dopaminergic potential shortly after expansion in the presence of FGF2. See Studer et al., Nat. Neurosci. Vol. 1:290-295 (1998). Similarly, human embryonic stem cell (ESC)-derived neural progenitors retain their positional identity, as determined by homeodomain transcription factor expression, and a high degree of neurogenic potential even after months of expansion. See Zhang et al., J. Hematother. Stem Cell Res. Vol. 12:625-634 (2003). The potential to produce large projection neurons such as midbrain dopamine neurons, spinal cord motor neurons, and hindbrain serotonergic neurons, however, fades within two to four passages and is replaced by other neuronal populations. This phenomenon creates a barrier for producing consistent populations of neuronal progenitors with predictable differentiation potential and functional properties. Accordingly, there remains a need for compositions and methods for expanding neuronal progenitors while maintaining the differentiation potential of the progenitors to yield the predicted array of diverse neuronal subtypes.